66 research outputs found
RNA–protein binding kinetics in an automated microfluidic reactor
Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome
The spread of mosquito-borne viruses in modern times:a spatio-temporal analysis of dengue and chikungunya
Comparative Phylogeography of a Coevolved Community: Concerted Population Expansions in Joshua Trees and Four Yucca Moths
Comparative phylogeographic studies have had mixed success in identifying common phylogeographic patterns among co-distributed organisms. Whereas some have found broadly similar patterns across a diverse array of taxa, others have found that the histories of different species are more idiosyncratic than congruent. The variation in the results of comparative phylogeographic studies could indicate that the extent to which sympatrically-distributed organisms share common biogeographic histories varies depending on the strength and specificity of ecological interactions between them. To test this hypothesis, we examined demographic and phylogeographic patterns in a highly specialized, coevolved community – Joshua trees (Yucca brevifolia) and their associated yucca moths. This tightly-integrated, mutually interdependent community is known to have experienced significant range changes at the end of the last glacial period, so there is a strong a priori expectation that these organisms will show common signatures of demographic and distributional changes over time. Using a database of >5000 GPS records for Joshua trees, and multi-locus DNA sequence data from the Joshua tree and four species of yucca moth, we combined paleaodistribution modeling with coalescent-based analyses of demographic and phylgeographic history. We extensively evaluated the power of our methods to infer past population size and distributional changes by evaluating the effect of different inference procedures on our results, comparing our palaeodistribution models to Pleistocene-aged packrat midden records, and simulating DNA sequence data under a variety of alternative demographic histories. Together the results indicate that these organisms have shared a common history of population expansion, and that these expansions were broadly coincident in time. However, contrary to our expectations, none of our analyses indicated significant range or population size reductions at the end of the last glacial period, and the inferred demographic changes substantially predate Holocene climate changes
Learning classification models of cognitive conditions from subtle behaviors in the digital Clock Drawing Test
Fluorescence Triple Correlation Spectroscopy Resolves Ten Intermediates Along Different Parallel Ribosome Assembly Pathways
The Spectroscopic Basis of Fluorescence Triple Correlation Spectroscopy
We have developed fluorescence triple correlation spectroscopy
(F3CS) as an extension of the widely used fluorescence microscopy
technique fluorescence correlation spectroscopy. F3CS correlates three
signals at once and provides additional capabilities for the study
of systems with complex stoichiometry, kinetic processes, and irreversible
reactions. A general theory of F3CS was developed to describe the
interplay of molecular dynamics and microscope optics, leading to
an analytical function to predict experimental triple correlations
of molecules that freely diffuse through the tight focus of the microscope.
Experimental correlations were calculated from raw fluorescence data
using triple correlation integrals that extend multiple-tau correlation
theory to delay times in two dimensions. The quality of experimental
data was improved by tuning specific spectroscopic parameters and
employing multiple independent detectors to minimize optoelectronic
artifacts. Experiments with the reversible system of freely diffusing
16S rRNA revealed that triple correlation functions contain symmetries
predicted from time-reversal arguments. Irreversible systems are shown
to break these symmetries, and correlation strategies were developed
to detect time-reversal asymmetries in a comprehensive way with respect
to two delay times, each spanning many orders of magnitude in time.
The correlation strategies, experimental approaches, and theory developed
here enable studies of the composition and dynamics of complex systems
using F3CS
Voting in professional associations: The case of the American sociological association revisited
Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy
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